Liquid chromatography is a basic separation technique that has been well established for chemical, biological, biochemical, environmental, and other analyses.
There are many principles of liquid chromatographic separation modes that have been known. Commonly, normal phase adsorption, reverse phase, ion exchange, or size exclusion modes are employed, but usually a single separation mode among these can be used successfully for liquid chromatographic analysis. If two or more separation modes could be combined orthogonally, a power of multiple modes of separation could be applied to a complex sample mixture.
Generally speaking, one liquid chromatographic system has a single pathway or mechanism for mobile phase control. Thus, when two or more different kinds of solid phase columns (after this, xe2x80x9ccolumnxe2x80x9d) are used, they are limited to a single mobile phase, or one kind column is used with a multiple selection valve for mobile phases. Alternatively, the analytes separated and eluted from a 1st column are collected when they elute. Subsequently, these are re-injected into 2nd system combined with a 2nd column using a 2nd mobile phase in a batch-wise process. If the mobile phase from the first separation is incompatible with the second column, an intermediate step, such as desalting or concentration, is implemented.
In the case of biological or clinical samples, the sample matrix is usually very complex.
Batch or two-step sample-collection makes it difficult to implement an automated separation system, and adds the disadvantages such as the loss of the analytes during transfer and the inconvenience of batch processing.
Using a combination between independent multiple systems based on orthogonal separation modes (such as ion exchange mode vs. reverse phase mode), it may be expected that the utilization of the different selectivity between target analytes and matrix contaminants will produce a much better separation. Because liquid chromatographs have only a single liquid flow path, it is necessary that multiple orthogonal systems be combined with columns and mobile phases integrated into one liquid chromatograph system.
Liquid chromatograph systems that have at least two orthogonal systems combined with columns and mobile phases are disclosed in several cited papers as examples.
For example, a first reference discloses a system in which analytes eluted from a 1st analytical column are trapped in two small volumes of sample tube on a switching valve (G. J. Opiteck et al., Anal.Chem. 69 (1997) 1518-1524). These sample tubes are alternately interchanged, trapping from a fraction from the 1st column and depositing it onto a 2nd analytical column. In this technique, the dead volume of the sample tube for trapping causes deleterious effects for separation at the 2nd column. Furthermore, desalting cannot be performed because no trapping column is used.
The second reference discloses a technique using a single trapping column for improved biological analysis (A. T. Davis et al., J. Chromatogra. B 752 (2001) 281-291). In this reference, only three elution bands (such as flow through, starting load, bound on 1st column) were used. Thus, separation on the 1st column may not enough for most of the analytes if there were multiple fractions. Also each of three bands was trapped just before each of the 2nd dimension analysis. Even if more than three bands can be separated on 1st dimension side, delivery of 1st mobile phase needs to be stopped while 2nd analysis is performing in order to prevent from mis-eluting to the waste and losing the analytes tapped on the 1st column. Further desalting using different solvent from 1st mobile phase cannot be performed in this system configuration.
Two similar techniques are disclosed in the third and fourth references (K. Wagner et al., J. Chromatogr. A 893 (2000) 293-305) and (G. J. Opiteck et al., Anal.Biochem. 258 (1998) 349-361). In both of these references, the eluent from the 1st column flows onto the 2nd column directly. Both systems alternate between two parallel separate 2nd columns mounted onto column switching valves, and switch between trapping and separating. Because the 2nd columns are used for both trapping and for a 2nd dimension separation, the differences between column properties can be difficult to balance, negatively affecting the results, and decreasing reproducibility. Also, each 2nd column presents a high backpressure for 1st column. High backpressure may reduce the lifetime and performance of the 1st column.
A fifth report discloses using 1st column and 2nd columns connected serially. Both 1st mobile phase and 2nd mobile phase are sent individually into both 1st and 2nd columns (A. J. Link et al., Nat. Biotechnol. 17 (1999) 676-682). This system does not have independent paths for the 1st and 2nd systems.
One common disadvantage among these reports is that desalting could not be performed before loading the analytes into a 2nd column when the effluent from 1st column requires salt containing buffers. Many choices for a second analytical chromatographic mode are incompatible with salt buffers for optimal separation. Additionally, because mass spectrometry is frequently used as a detector to provide sensitivity and selectivity, the samples (or solutions) containing non-volatile salts are incompatible with optimal performance. Deposition of salt interferes with electrospray ionization and transfer of the vaporized ions into the mass spectrometer.
References are also given for the equipments, parts and techniques, which this invention utilizes:
The catalog of 14 port rotary valve (Malco Instruments Co. Inc., TX)
The catalog of LC-VP series (Shimadzu Corporation, Japan)
The catalog of CapTrap as used trapping column (Michrom BioResources, Inc., CA)
In view of the problem described above, the object of the, present invention is to provide a multi-dimensional liquid chromatograph separation system that can perform automatic separations of samples containing complex mixtures.
A liquid chromatograph separation system according to the present invention that has properties includes at least two or more individual systems. Each of the systems has a mobile phase and a column and controls independently the mobile phase that flows through the column. The system has a plurality of trapping columns for trapping analytes with the mobile phase that are eluted from the column. In addition, the system has a mechanism for selecting either loading the analytes eluted from the column onto the trapping columns, or diverting the mobile phase to waste, and a mechanism for eluting the analytes trapped on each trapping column and for online loading onto a second analytical column.
In another aspect of the present invention, the liquid chromatograph system further comprises a system for detection of separated analytes eluted from the second column or a last column if there is a series of more than two systems with more than two columns.
In further aspect of the present invention, the liquid chromatograph system further comprises a system for detection of separated analytes eluted from the column or an intermediate column if there are more than two independent systems and columns.
In still further aspect of the present invention, the liquid chromatograph system further comprises a system for desalting that is set up independently from any other systems. The desalting is performed after trapping the analytes on each trapping column and before loading onto the next column, and a solvent for desalting is different from those of any other mobile phase and mobile phase.
Finally, all of these processes including injection and desalting process are performed continuously online without attendant, and uninterrupted. Many samples can be analyzed routinely and successively using this system. This provides an economic advantage by increasing through-put for complex mixture analyses using automation.